Fluorescence technology is enjoying ever-increasing interest from many areas of science including chemistry and biology. In certain instances, fluorescent molecules can be used to detect the presence or absence of analytes or chemicals in food and environmental samples. Some sensitive and quantitative fluorescence detection devices are required for in vitro biochemical assays such as DNA sequencing and blood glucose quantification. In some instances, fluorescent probes having suitable photochemical properties can be used for tracing molecular and physiological events in living cells as well as for high-throughput screenings. In general, fluorescence technology is simpler and can provide higher sensitivity and more molecular information than other types of optical measurements. Fluorescence measurements are generally highly sensitive because of the low level of fluorescence background generally found in most chemical and biological samples. Along with the recent advances in fluorescence instrumentation such as confocal and multi-photo fluorescence microscopies, three-dimensional imaging of cellular events and biological species dynamics have become possible in real-time.
Despite of the above-mentioned advantages, the feasibility of using fluorescence technology for a particular application can often be limited by the availability of appropriate fluorescent molecules. Fluorescent molecules generally include fluorescent label (or fluorescent tracer) molecules and fluorogenic probe (or fluorogenic sensor) molecules, both of which differ in their fluorescence properties. In general, the fluorescence of fluorescent label molecules always keeps in “on” state. The fluorescent label molecules may be used to track biological species. On the contrary, the fluorescence properties of the fluorogenic probe molecules can be changed by bonding to or reacting with certain analytes or chemicals. Therefore, the fluorogenic probe molecules can be used to trace, measure, detect or screen various analytes or chemicals that bond or react with the fluorogenic probe molecules.
When used as fluorescent labels or fluorescent dyes, the fluorescent label molecules need to chemically react with a biologically active target, such as a protein, DNA, enzyme, antibody, organelle, cell, tissue, drug, hormone, nucleotide, nucleic acid, polysaccharide, lipid or other biomolecules, to yield a fluorescent derivative of the target. The number of available fluorescent label molecules that can react with a biologically active target is generally limited. Reactive fluorescein and rhodamine derivatives have been by far the most widely used fluorescent label molecules because they can be suitably excited by commonly used light sources, such as the argon laser (488 nm and 514 nm lines) and the mercury arc lamp (546 nm line). However, they have some undesirable properties that may preclude them to be used in some useful applications. For example, fluorescein derivatives can be photo-bleached in a relative high rate and may show pH dependent absorptions that lead to significantly reduced fluorescence at or below physiological pH value 7. On the other hand, rhodamine derivatives, which are relatively photostable and exhibit pH insensitive fluorescence compared to fluorescein derivatives, show much lower quantum yields in aqueous solution and further decreases in quantum yield when conjugated with proteins. Furthermore, the low water solubility of rhodamine derivatives and conjugates thereof presents difficulties for the preparation and use of fluorescent label molecules comprising rhodamine derivatives.
In view of the undesirable properties of the fluorescein and rhodamine derivatives mentioned above, there is a need for new fluorescent label molecules that have improved properties over those of fluorescein and rhodamine derivatives. Some rhodol or fluorescein derivatives, such as those described in Ioffe et al., J. Org. Chem. USSR 1965, 1, 326-336; Reynolds, G. A., U.S. Pat. No. 3,932,415; Lee et al., Cytometry 1989, 10, 151-164; Haugland et al., U.S. Pat. No. 5,227,487; Whitaker et al., Anal. Biochem. 1992, 207, 267-279, may be used as alternative fluorescent label molecules because they generally have the desirable properties of fluorescein and rhodamine. For example, they have desirable absorption properties, low sensitivity of fluorescence to pH in the physiological range and solvent polarity, high extinction coefficients, high quantum yields, high photostability, and high solubility in a variety of solvents.
Despite their desirable properties, the applications of rhodol fluorescent dyes as fluorescent labels or fluorogenic probes are seldom reported in the literature, which may be due to difficulties of synthesizing rhodol compounds. In general, a key synthetic step of making a rhodol fluorophore is condensing a substituted or unsubstituted resorcinol with a substituted or unsubstituted 6-acyl-3-aminophenol in the presence of a Lewis acid catalyst such as zinc chloride or a dehydrating acid. Alternatively, the rhodol fluorophore can be prepared by condensing a substituted or unsubstituted amino phenol with a substituted or unsubstituted 4-acyl-resorcinol in the presence of a Lewis acid catalyst. The general synthetic methods mentioned above generally suffer from low yield and tedious purification work. Therefore, there is a need for developing a more efficient synthetic method for making fluorophore compounds such as rhodol fluorophores.
Recently, the developments of fluorogenic probes for different targets have encountered a bottleneck. It may be due to the fact that most currently available fluorogenic probes are developed empirically, but not rationally, which can hamper the design of new fluorogenic probes. Although there have been some reported methods of applying theoretical approaches to the design of fluorogenic probes, these are still far from enough. Another effective approach involves combinatorial synthesis of libraries of fluorogenic probe molecules. Rhodol fluorophores are suitable candidates in a fluorogenic probe library because of their advantageous photophysical properties as stated above. Therefore, there is also a need for a library of fluorophore compounds including rhodol fluorophores that can be easily developed by the new and efficient synthetic methods for making fluorophore compounds disclosed herein.